Immunoblot analysis of BST-2 glycosylation mutants. (A) 293T cells were transfected with wt BST-2, single glycosylation site mutants N1 & N2, or the double glycosylation site mutant N1/N2. BST-2 specific proteins were identified by immunoblotting using a BST-2-specific polyclonal antibody (top panel). Aliquots of the same samples were adsorbed to either ConA (middle panel) or DS lectin (lower panel) as described in Methods. Eluates were analyzed by immunoblotting using a BST-2-specific polyclonal antibody. (B) 293T cells were transfected with 5 μg each of NL4-3 wt (lanes 1-4) or NL4-3/Udel (lanes 5-8) either in the absence of BST-2 (lanes 1 & 5) or in the presence of 0.01 μg (lanes 2 & 6), 0.03 μg (lanes 3 & 7), or 0.1 μg (lanes 4 & 8) BST-2 DNA. Cells were harvested 20 h post transfection. A fraction of the cells was used for immunoblot analysis; the other part was used for metabolic labelling (Fig. 4). Whole cell lysates were prepared and used for immunoblot analysis using a BST-2-specific polyclonal antibody (top two panels). The blots were then sequentially reprobed with antibodies to Vpu (third panel) or tubulin (lower panel). Representative samples shown in the lower panels were from the N1/N2 blot. Proteins are identified on the right. The arrow points to a form of BST-2 N1/N2 whose migration in the gel is consistent with a dimer.